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INDM 3007

INDM 3007. Lecture 4. F-6-P. Xu5P. F-1,6-P. GAP. DHAP. GAP. E-4-P. DHAP. 1,3-PGA. S-1,7-P. 3-PGA. S-7-P. Calvin cycle. 2-PGA. R5P. Xu5P. CO. PEP. 2. Ru-1,5-P. Ru-5-P. Pyruvate. CO. 2. AcetylCoA. oxaloacetate. citrate. TCA. cycle. RuBisCO. 300. PRK. Hydrogenase.

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INDM 3007

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  1. INDM 3007 Lecture 4

  2. F-6-P Xu5P F-1,6-P GAP DHAP GAP E-4-P DHAP 1,3-PGA S-1,7-P 3-PGA S-7-P Calvin cycle 2-PGA R5P Xu5P CO PEP 2 Ru-1,5-P Ru-5-P Pyruvate CO 2 AcetylCoA oxaloacetate citrate TCA cycle

  3. RuBisCO 300 PRK Hydrogenase 200 Enzyme activity (nmol/min/mg protein) 100 0 H2/CO2 Succinate Succinate/H2 Gluconate/H2

  4. cbboperon: induced during autotrophic growth + cbbR cbbL cbbS cbbX cbbF cbbP cbbT cbbA cbbE + gap-pgk operon: super-induced during autotrophic growth gap pgk Genetic organisation of the Calvin cycle

  5. RegA regulon genA genD Operon Regulon: Operons with related metabolic function that share a common regulator

  6. cbb operon cbbR CbbR is a LysR-type regulator Protein family: group of proteins sharing sequence similarity common evolutionary origin have common function (often not always!) e.g, transcriptional regulator

  7. ‘lay-out’ of LysR type protein NH2 COOH DNA binding domain Effector binding domain Multimerisation domain LysR type regulator • Transcriptional activators (and repressors) • second most abundant group of regulators, occur in all bacteria • Control wide range of functions, ie virulence, antibiotic production etc • almost 200 proteins known • Molecular mass usually around 36 kDa Note: every LysR-type regulator binds to different DNA sequence and recognises different ligand

  8. kDa 175 83 62 48 33 Denaturing conditions: 36 kDa 25 Non denaturing condtions: 79 kDa CbbR is a dimer in solution 17 7 Expression and purification of CbbR Strong promoter cbbR

  9. Label DNA with radioactive nucleotides Incubate protein with DNA Analyse mixture on acrylamide gel DNA DNA+protein Negative Positive Analysis of DNA-Protein interaction Gel retardation or band-shift assay

  10. cbbL cbbR _ + CbbR Complex 1 Complex 2 Free fragment Interaction between CbbR and the cbb promoter

  11. Label the end of one DNA strand Incubate with protein Digest DNA at random with DNaseI Analysis of DNA-Protein interaction Footprinting Trick: Dnase I will digest anywhere, but NOT where protein is bound. This is protected!!

  12. Denature DNA, and load on gel DNA DNA+protein Protected region: footprint DNA:DnaseI ratio such that every DNA molecule is only digested once

  13. cbbL ATGATGGCGGCCCTCCTGTTTGTTGAGGTGACCTTGCCCCCGCGCCG cbbR ATTTC AGGTAAATTTAAATGA TATGA AGTAGGCATTCAGGAAATCTGAAGTG * cbbR Cbb operon Conclusion: two cbbR dimers bind side by side cbb operon mRNA IR3 IR2 IR1

  14. Effect of spacing between IR1 and IR2/3 on cbb promoter activity IR1 IR2 IR3 GCCACTTCAGATTTCCTGAATGCCTACTTCATATCATTTAAATTTACCTGAAATCGGCGCGGGGGCA Spacing (nt) Activity (%) 5 0 GC CT 6 0 GC CT 10 66 GC CT CT 12 0 GC 14 0 GC CT

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